Seroprevalence and associated risk factors of leptospirosis in bovine dairy farms in Telangana state, India.

The current cross-sectional study aimed to determine the seroprevalence of Leptospira infection in bovine dairy farms in the Telangana state of India, as well as the associated risk factors, in order to implement effective preventive measures for disease control. A total of 469 blood samples were collected from 67 herds/farms in different areas, covering 20 administrative districts in the state. These samples consisted of 253 from cattle and 216 from buffaloes. Questionnaires were used to collect data on host and epidemiological factors. The collected sera were tested using the gold standard serological test, the Microscopic Agglutination Test (MAT), which employed a panel of 18 reference serovars for Leptospira exposure. The statistical analysis of epidemiological data was carried out to identify the risk factors associated with Leptospira exposure. The overall observed seroprevalence at the animal and farm levels was 41.4% and 77.6%, respectively. The most prevalent anti-leptospiral antibodies were observed against the serogroups Icterohaemorrhagiae (32.4%), Pomona (22.2%), Javanica (19.1%), Australis (17.0%), Bataviae (15.5%), Autumnalis (12.9%), Hebdomadis (12.9%), and others, in the total reacting samples. At the animal level, the significant risk factors associated with exposure to Leptospira species were breed (p = 0.03) and health status (p = 0.03). Furthermore, the multivariate statistical analysis of farm factors revealed that farm size (p = 0.05), presence of dogs (p = 0.04) and rodents (p = 0.01) on the farm, use of fodder from wet soils (p = 0.04), and proximity to water bodies (p = 0.04) were significantly associated with exposure to Leptospira in the studied region. This study provides the first report from India highlighting the important risk factors at the herd/farm and animal level associated with Leptospira infections in cattle and buffaloes. The findings contribute to strengthening the one-health strategy by facilitating the design and planning of appropriate control measures to alleviate the burden of leptospirosis in bovines.

Prevalence of Leptospira serogroup-specific antibodies in cattle associated with reproductive problems in endemic states of India.

In this study, the seroprevalence and distribution of Leptospira in dairy cattle in endemic states of India were investigated in association with reproductive problems of the cattle. A total of 373 cattle serum samples from 45 farms in Maharashtra, Gujarat, Andhra Pradesh, Telangana, Karnataka, Tamil Nadu, Punjab, Haryana, Chhattisgarh, Sikkim and Uttarakhand states were collected from animals with a history of reproductive disorders like abortion, repeat breeding, anoestrus and endometritis, and also from apparently healthy animals. These samples were screened for Leptospira serogroup-specific antibodies by microscopic agglutination test (MAT) using a panel of 18 live reference serovar antigens. The seropositivity of 70.51% (263/373, 95% CI 0.65 to 0.75) was associated with reproductive problems (χ2 = 55.71, p < 0.01) and sampled states (χ2 = 32.99, p < 0.01) and independent of apparently healthy animals (χ2 = 15.6, p > 0.10) and age groups of cattle (χ2 = 0.91, p > 0.10). Further, the odds (risk-relation) of reproductive disorders was 5.29 compared to apparently healthy animals (0.25 odds). The frequency distribution of predominant serogroup-specific Leptospira antibodies were determined against the serovars: Hardjo (27.76%), Pyrogenes (18.63%), Canicola and Javanica (17.49%), Hebdomadis (17.11%), Shermani and Panama (16.73%), Djasiman (16.35%), Tarassovi, Grippotyphosa and Pomona (15.97%), Icterohaemorrhagiae (15.59%), Copenhageni (14.83%), Australis (13.69%), Kaup and Hurstbridge (10.65%), Bankinang (10.27%) and Bataviae (9.51%). In conclusion, dairy cattle have a role in maintaining important several serovars besides well-known Hardjo serovar in endemic states of India and warrant mitigating measures to reduce the incidence of cattle leptospirosis including need for an intensive surveillance programme, preventive vaccination and control strategies.

Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

Molecular typing of Brucella species isolates from livestock and human.

Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus-melitensis-ovis-suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.

Comparative evaluation of the immunodominant proteins of Brucella abortus for the diagnosis of cattle brucellosis.

BACKGROUND AND AIM: The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis. MATERIALS AND METHODS: Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done. RESULTS: All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden's index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85). CONCLUSION: BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.

Spatial seroprevalence of bovine brucellosis in India-A large random sampling survey.

Brucellosis caused by Brucella spp. is an important zoonosis and constitutes a serious public health hazard. In India, the disease is increasingly prevalent among bovine population with high zoonotic potential and negative impact on national economy. The investigation was conducted to study seroprevalence of brucellosis through random sample survey using survey tool box software. A total of 12,054 [cattle-9236, buffaloes-2818] bovine serum samples sourced from 15 states of India were tested by protein G indirect ELISA. The true prevalences of brucellosis observed in cattle and buffaloes were 8.3% and 3.6%, respectively. The highest prevalence of brucellosis was observed in the state of Punjab in both cattle and buffaloes (23.51 and 10.2%). Comparatively higher prevalence was recorded in cattle than the buffaloes in all the states except Manipur. The true prevalence greater than 5% was recorded in 8 and 3 states for cattle and buffaloes, respectively [(cattle- Punjab, Maharashtra, Rajasthan, Karnataka, Madhya Pradesh, Tamil Nadu, Gujarat and Kerala) and (buffaloes-Punjab, Gujarat and Manipur)] indicating wider prevalence of brucellosis. This study conclusively highlighted the seroprevalence of bovine brucellosis at state level which might be useful for prioritizing regions for vaccination, designing control strategies and improvisation of clinical surveillance system.